1. IN VITRO EMBRYO PRODUCTION Dr. R. A. Siddique M.V.Sc. PhD Scholar N.D.R.I. Karnal (Haryana) INDIA

2. In vitro embryo production (IVEP ) Involves: Collection of oocytes Collection of oocytes In vitro maturation (IVM) In vitro maturation (IVM) In vitro fertilization (IVF) In vitro fertilization (IVF) In vitro culture (IVC) In vitro culture (IVC)

3. Collection of oocytes Live animal Ovum pick-up Slaughtered animal Aspiration

4. Collection of oocytes ovaries collected from slaughterhouse Transport to lab in saline containing antibiotics at 32-37  C Aspiration of follicles (2-8 mm) in aspiration medium (TCM ‑ 199 + 10% FBS)

5. Grading of oocytes Grade A:Compact cumulus ‑ oocyte ‑ complexes (COCs) with an unexpanded cumulus mass having ≥4 layers of cumulus cells, and with homogenous evenly granular ooplasm Grade B:COCs with 2-3 layers of cumulus cells and a homogenous evenly granular ooplasm Grade C: Oocytes partially or wholly denuded or with expanded or scattered cumulus cells or with an irregular and dark ooplasm

6. In vitro fertilization Frozen semen Thaw In vitro sperm treatment and Capacitation In vitro matured oocytes Incubation with 1-2 million/ml spermatozoa for 18 h Presumptive zygotes washed with IVC media

7. Sperm treatment and Capacitation Through washing and exposure in media Through washing and exposure in media Enhance motility of spermatozoa Enhance motility of spermatozoa Express the acrosome reaction Express the acrosome reaction Enhance successful fertilization of the oocytes Enhance successful fertilization of the oocytes

8. Components of Capacitation media  Bovine serum albumin  Heparin - glucoseaminoglycans  Caffeine – cyclic nucleotide phosphodiesterase inhibitor

9. In vitro culture of embryos Presumptive zygotes washed with IVC media Placed in 50-100 μl droplets of IVC media in 35 mm Petri dish in groups of 10-15 Cultured for 9 days in IVC medium in a CO 2 incubator (5% CO 2 in air,90-95% humidity) at 38.5°C

10. Important consideration for IVMFC Water quality Water quality Buffering system and Osmolarity Buffering system and Osmolarity Flux culture Flux culture Effect of maturation time Effect of maturation time

11. Other consideration..  Antibiotic cover & mineral oil covering  Light environment  Temperature and gas phase  Storage and maintenance of media

12. Culture supplements Protein Hormones and growth factors Culture vessel

13. In vitro culture system Complex medium with somatic cells Simple medium

14. Culture in complex media  TCM-199  F-10  MEM  RPMI  Oviductal epithelia cells  Granulosa cell  VERO  BRL  STO  3T3 Somatic cells

15. Culture in simple media mCRaa1 mCRaa2 mSOFaa KSOMaa G1/G2 No somatic cells

16. Criteria to evaluate the quality of embryo Excellent : Spherical,Symmetrical with cells of uniform size Excellent : Spherical,Symmetrical with cells of uniform size Good : Few extruded blastomeres, irregelar shape Good : Few extruded blastomeres, irregelar shape Fair : Extruded blastomeres, few degenerated cells Fair : Extruded blastomeres, few degenerated cells Poor : Numerous extruded blastomeres, degenerated cells Poor : Numerous extruded blastomeres, degenerated cells

17. Evaluation of embryo development 2-16 cell stages 2-16 cell stages Morula : Embryo with >32 cell stage Morula : Embryo with >32 cell stage Compact morula : Compaction of blastomeres Compact morula : Compaction of blastomeres Early blastocyst : Formation of blastocoel had just started Early blastocyst : Formation of blastocoel had just started Blastocyst : Well definrd blastocoel & start differentiation Blastocyst : Well definrd blastocoel & start differentiation Expanded blastocyst : Expanded with zona thinning Expanded blastocyst : Expanded with zona thinning Hatched blastocyst : partly or completely come out from ruptured zona Hatched blastocyst : partly or completely come out from ruptured zona

18. Chronology of embryo development Cell stage Estimated age in days Cattlebuffalo Morula54 Compact morula 65 Early blastocyst 76 Blastocyst76 Expanded blastocyst 87 Hatched blastocyst 9 7.5 to 8

19. Application of IVEP Stem cellTransgenic animal Cloning Cryopresevation Embryo sexingDrug testing Early developmental gene Genome reprogramming